Our goal is to elucidate the signaling pathway involved in sperm hyperactivation. Hyperactivation, a vigorous type of sperm motility, is found in the oviduct at time of fertilization. Because the ability of sperm to hyperactivate is often an indictor of sperm fertility, determining the mechanism by which hyperactivated motility is regulated can lead to development of treatments to promote sperm fertility or alternatively, result in development of contraceptives targeting specific modulators of sperm motility. Our specific aims are to use procaine-induced hyperactivated bull sperm to determine the role of: 1) Protein phosphorylation in hyperactivated sperm using western blot analysis and indirect immunofluorescence. 2) Intracellular Ca 2+ in hyperactivated motility using fluorescent Ca 2+ indictors and single cell imaging. 3) Cytoplasmic alkalization in modulating hyperactivated motility by measuring intracellular pH using pH sensitive fluorescent probes and single cell image analysis. We propose that hyperactivation is mediated by a signaling pathway involving phosphorylation of flagellar proteins, which is dependent on Ca 2+ influx and alkalization of the cytosol. [unreadable] [unreadable]